The long-term goal of this proposal is to study mechanism(s) of pathogenicity of Cryptococcus neoformans (Cn) focusing on how Cn sphingolipids regulate fungal virulence and counteract the host immune response. Cn is a fungal pathogen that, upon entering the lung and disseminating through the bloodstream, causes a life- threatening meningo-encephalitis in susceptible patients. One key feature of Cn is its ability to grow both extracellularly and intracellularly. Being the lung the port of entry of Cn, alveolar macrophages (AMs) are the first line of defense of the host. Thus, the outcome of the interaction between the fungus and AMs greatly determines the progression of the disease. If the immune system of the host is compromised and AMs fail to kill the engulfed Cn cells, these fungal cells survive and proliferate within the phagolysosome of AMs. This implies that the physiopathology of cryptococcosis is determined by fungal growth that can occur in an intracellular compartment (e.g. acidic environment of AM's phagolysosome) and in an extracellular compartment (e.g. neutral/alkaline environment of alveolar spaces and bloodstream). In the past funding cycle, we identified novel fungal factors in the sphingolipid pathway that specifically regulate growth of Cn in each compartment. Particularly, the inositol phosphosphingolipid phospholipase C (Isc1) enzyme was found to be required for intracellular growth of Cn within AMs, whereas the glucosylceramide synthase (Gcs1) was found to be required for extracellular growth of Cn within the alveolar spaces. Importantly, we identified very long chain C26 phytoceramide and glucosylceramide (GlcCer) as the biochemical products of the reactions catalized by Isc1 and Gcs1, respectively. Thus, we hypothesize that Isc1 and Gcs1 regulate pathogenicity by favoring growth in different compartment(s) through the action of the specific sphingolipids that they produce. This hypothesis will be addressed by the following Aims: 1) To study the mechanism by which fungal sphingolipids regulate growth of Cn in the intracellular compartment; 2) To study the mechanism by which fungal sphingolipids regulate growth of Cn in the lung-like extracellular compartment; 3) To determine the therapeutic effect of targeting intracellular and/or extracellular populations of Cn on the outcome of the infection.